1. Introduction
3.3 Results
3.3.1 The rate of environmental change regulates cellular phenotype
We compared cell viability, cell signaling, and metabolism in cells exposed to either linear (ramp) or acute (step) concentration changes in the environments in which the final concentration and the total amount of osmotic stress (Area Under the Curve - AUC) is identical (Figure 3.1a, Figure 3.2). We identify the dynamic range of cell viability by determining the tolerance of monocytes (THP1 cell line, male, acute monocytic leukemia), T-cells (Jurkat, male, acute T cell leukemia), and cervical cells (HeLa, female, cervical adenocarcinoma (Dmitrieva and Burg 2008)) to step increases in NaCl concentrations (Figure 3.1b). In the non-stress control condition, cells grow in culture under physiological NaCl concentrations of about 280 mosmol/l NaCl to which we add the hypertonic osmolytes NaCl and mannitol. To stress the cells and mimic in vivo osmolyte changes, we add up to 400 mosmol/l NaCl to the cells (Figures 3.1, Figure 3.3). We observe that the viability decreases with an increased NaCl concentration of up to 300 mosmol/l. At and above of 300 mosmol/l NaCl, the viability is below 15% for all cell lines. Our results in the abovementioned cell lines are consistent with previous studies in HeLa cells (Figure 3.1b) (Dmitrieva and Burg 2008), indicating that different cell types respond similarly to hypertonic stress.
Figure 3.1: Human cell fate decisions are regulated differently upon step or ramp treatment conditions.
a) Kinetic environments such as concentration ramps, as observed in different physiological relevant conditions, may differentially modulate cell signaling, cell fate, and phenotype even if the final concentration and total amount of stress are identical. Step experiments finish earlier than ramp experiments to account for the same total exposure or Area Under the Curve (AUC). b) We measured relative cell viability after exposure to instant hyperosmotic stress (NaCl for 5h for Jurkat, THP1) or 24h (HeLa cells). Cell viability was determined by measuring intracellular ATP (Jurkat, THP1) or cell counts (HeLa). The shaded area represents the standard deviation (SD) (Jurkat, THP1) or Standard Error (SE) (HeLa) (25) c,d) Relative cell viability was determined for step (c,d) and 10h ramp (d) treatment (see insert) after addition of (c) 300 mosmol/l or (d) 200 and 400 mosmol/l osmolyte. We determined viability at the end of the experiment after reaching the same cumulative exposure of additional NaCl. Bars represent data from at least 3 independent experiments for each condition. Error bars represent SD. two-sided unpaired student’s t-test: **p<0.01, ***p<0.001, ****p<0.001.
Figure 3.2: Kinetic environment input profiles applied for different step and ramp treatments.
Black lines represent the change of added NaCl osmolarity concentration over time. Cells exposed to a step or 3 and 6h ramp treatment are incubated at the final NaCl concentration until the cumulative exposure is identical between the different conditions.
Figure 3.3: Viability improvement in hypertonic stress during a ramp vs. a step is a general cell biological feature independent of the cell line or osmolyte.
a) Viability for THP1 cells measured by intracellular ATP for step addition of NaCl (0), and ramps of 3,4,5,6,10h to indicated concentrations in mosmol/l. We determine viability at the end of the experiment at the same cumulative exposure of additional NaCl. Boxplots represent data from at least 3 independent experiments for each condition. b,c) Viability for Jurkat cells by measuring intracellular ATP for instant addition of mannitol (0), and ramps of 3,6,10h to indicated concentrations in mosmol/l for mannitol (b) and NaCl (c).
We determine viability at the end of the experiment at the same cumulative exposure of additional mannitol. Boxplots represent data from at least 3 independent experiments for each condition.
We then quantified the response of different cell lines (Jurkat and THP1) to different rates and final NaCl concentrations (Figure 3.1c-d, Figure 3.3). To compare the different conditions for the same final NaCl concentration, we exposed cells to the same cumulative exposure by integrating the total amount of NaCl over the entire profile (AUC).
We perform experiments for each NaCl concentration for ramp durations of up to 10h.
For experiments with ramp durations of less than 10h, cells stay at the final NaCl concentration until the AUC is identical to the 10h ramp experiment (Figure 3.2). When we expose Jurkat cells to 300 mosmol/l hypertonic osmolyte, the viability improves from 15% to 40% for a ramp duration of 10h (Figures 3.1c,(black), Figure 3.3c cyan). In comparison, a step increase of 200 mosmol/l NaCl to the media for 5h reduces viability to around 50% and shows only minor improvement with increases in ramp duration (Figures 3.1d (black), Figure 3.3c, magenta). For the step condition of added 400 mosmol/l NaCl for 5 h, the viability is below 5% and shows only minor improvement with increasing ramp durations (Figures 3.1d (black), Figure 3.3, green and yellow). These observations are consistent in THP1 cells, indicating that this effect is reproducible in a different cell line and cell type (Figures 3.1c-d (light grey), 3.3a). To distinguish the effect of cell viability between NaCl toxicity and changes in external osmolarity, we repeated the experiments with mannitol in the Jurkat cell line at the same osmolar concentrations (Figures 3.1c-d (dark grey), Figure 3.3b). Mannitol is not able to easily pass through the cell membrane and is known to have low cell toxicity. When we add 300 mosmol/l Mannitol to the medium, Jurkat cells survive better during the ramp compared to the step treatment. This comparison shows no difference between cells treated with NaCl or Mannitol, indicating extracellular hypertonicity and not NaCl-specific toxicity drive these effects. These results strongly suggest that cell viability improvements, while slowly increasing NaCl concentration, are a robust cell type- and cell line-independent hypertonic stress response. We further tested our hypothesis in human primary blood mononuclear cells (PBMCs). We observe very similar levels of viability after the step and 10h ramp treatments (Figure 3.4). However, there we observe more variability between the cells of the 3 different donors we tested than there is between Jurkat and THP1 cells which could indicate a different response to NaCl hypertonicity between different donors.
Figure 3.4: Viability in hypertonic stress during a ramp vs. a step in PBMCs.
a) Viability for THP1 cells measured by intracellular ATP for step addition of NaCl (0), and 10h ramps 300 mosmol/l. We determine viability at the end of the experiment at the same cumulative exposure of additional NaCl. Boxplots and symbols represent data from at 3 donors.
3.3.2 A functional temporal screen identifies regulators of cell viability in step and